Semi-synthesis of Mycolactone A/B and Conjugable Derivatives for Sec61 Binding Assays

Mycolactone A/B (MycA/B) is reportedly the sole virulence factor of Mycobacterium ulcerans which causes the neglected tropical disease Buruli ulcer. This disease causes tissue destruction and suppression of local inflammation. The mechanism of action of MycA/B involves inhibition of protein translocation by Sec61, a transmembrane pore which permits passage of polypeptide chains across or into endoplasmic reticulum (ER) membranes. We hypothesise that binding of mycolactone to Sec61 can be inhibited, by a small molecule, without affecting translocation. Discovery of such a molecule requires the development of an assay to test the extent to which mycolactone is binding to Sec61 and this is the aim of this project. The assay is based on Bioluminescence Resonance Energy Transfer (BRET) and requires:
1. Genetic modification of M. ulcerans to produce a Sec61-NanoLuciferase (NLuc) enzyme fusion protein which emits light through reaction of its substrate; and,
2. Chemical conjugation of a fluorophore to MycA/B to give a 'tracer' ligand.

When the MycA/B tracer binds to Sec61, the light emitted from proximal NLuc is absorbed by the fluorophore and emitted at a different, measurable wavelength. In the presence of inhibitors, there will be less emitted fluorescence in proportion to the concentration and binding strength of the inhibitor.
This PhD project focuses on synthesis of the MycA/B-fluorophore tracer. Literature syntheses require at least 17 steps for the MycA/B core, 17 for the fatty acid tail and 2-3 more for their coupling and deprotection. In addition, the slow and hazardous growth of M. ulcerans precludes isolation of MycA/B. Hence, a novel, efficient semi-synthesis of MycA/B will be developed along with variations to produce analogues amenable to conjugation with the fluorophore.

The proposed semi-synthesis firstly involves isolation of alternative mycolactone F (MycF, Figure 1), a fatty acid chain variant produced by the fish pathogen M. marinum. Since this is less pathogenic to humans and grows at a faster rate, its culture can be scaled up and MycF isolated in larger quantities.

Protection of the alcohols as TBS-ethers (Scheme 1) followed by hydrolysis of the enone ester should provide the core 1 with one free alcohol ready for coupling with synthetic MycA/B fatty acid side chain.

A convergent synthesis of the MycA/B fatty acid side chain (Scheme 2) will use Burke's iterative MIDA boronate methodology for the polyene 2 and chiral pool asymmetric synthesis of the triol precursor 3. Coupling of the two will be achieved in a stereoselective manner to give 4. Hydrolysis of the ester followed by attachment to the mycolactone core 1 by esterification will give alcohol-protected MycA/B in the shortest route to date.

With this route established, it will be modified to provide amine-functionalised MycA/B ready for conjugation to a fluorophore and the development of a Sec61-binding assay.

References
(1) Chany, A.-C.; Tresse, C.; Casarotto, V.; Blanchard, N. Nat. Prod. Rep. 2013, 30, 1527.
(2) Hall, B. S.; Ogbechi, J.; Simmonds, R. E.; Hill, K.; McKenna, M.; High, S.; Willis, A. E. PLoS Path. 2014, 10, e1004061.
(3) Ogbechi, J.; Hall, B. S.; Bodman-Smith, K.; Simmonds, R. E.; Ruf, M.-T.; Pluschke, G.; Vogel, M.; Wu, H.-L.; Stainer, A.; Esmon, C. T.; Ahnstrom, J. PLoS Pathog 2015, 11, e1005011.
(4) Schurmann, M.; Janning, P.; Ziegler, S.; Waldmann, H. Cell Chem. Biol. 2016, 23, 435.
(5) Brown, C. A.; Aggarwal, V. K. Chem. Eur. J. 2015, 21, 13900.
(6) Wang, G.; Yin, N.; Negishi, E.-i. Chem. Eur. J. 2011, 17, 4118.
(7) Ranger, B. S.; Mahrous, E. A.; Mosi, L.; Adusumilli, S.; Lee, R. E.; Colorni, A.; Rhodes, M.; Small, P. L. C. Infect. Immun. 2006, 74, 6037.
(8) Song, F.; Fidanze, S.; Benowitzx, A. B.; Kishi, Y. Tetrahedron 2007, 63, 5739.
(9) Woerly, E. M.; Roy, J.; Burke, M. D. Nat Chem 2014, 6, 484.